In Vitro Susceptibilities of Worldwide Isolates of Intrapulmonary Aspergillus Species and Important Candida Species in Sterile Body Sites against Important Antifungals: Data from the Antimicrobial Testing Leadership and Surveillance Program, 2017–2020

ABSTRACT To understand the changes of resistance in clinically commonly encountered fungi, we used the Antimicrobial Testing Leadership and Surveillance (ATLAS) database to explore in vitro antifungal susceptibilities against clinically important isolates of Aspergillus and Candida species (collected from intrapulmonary and sterile body areas, respectively). We applied the CLSI antifungal 2020 and the EUCAST antifungal 2020 guidelines. From 2017 to 2020, isolates of intrapulmonary Aspergillus fumigatus (n = 660), Aspergillus niger (n = 107), Aspergillus flavus (n = 96), Aspergillus terreus (n = 40), and Aspergillus nidulans species complex (n = 26) and sterile site-originated isolates of Candida albicans (n = 1,810), Candida glabrata (n = 894), Candida krusei (n = 120), Candida dubliniensis (n = 107), Candida lusitaniae (n = 82), Candida guilliermondii (n = 28), and Candida auris (n = 7) were enrolled in this study. Using the EUCAST 2020 breakpoints, it was demonstrated that amphotericin B and posaconazole displayed poor in vitro susceptibility rates against A. fumigatus isolates (<50% and 18.9%, respectively). In contrast, isavuconazole and itraconazole showed high in vitro potency against most Aspergillus isolates (>92%). Most intrapulmonary Aspergillus isolates exhibited MICs of ≤0.06 μg/mL to anidulafungin. Furthermore, intrapulmonary A. fumigatus isolates collected from Italy and the United Kingdom exhibited lower in vitro susceptibility to isavuconazole (72.2% and 69%, respectively) than those in the remaining ATLAS participant countries (>85%). Higher isavuconazole MIC90s against C. auris and C. guilliermondii (1 and 4 μg/mL, respectively) were observed compared to the other five Candida species. Despite the aforementioned MICs and susceptibilities against fungi, research needs to consider the pharmacokinetic (PK) profiles, pharmacodynamic (PD) parameters, and clinical treatment experience with antifungals against specific Aspergillus species. IMPORTANCE In addition to monitoring the antifungal susceptibilities of clinically important fungi, reviewing the PK/PD indices and the clinical therapy experience of antifungals under evaluation are important to guide an appropriate antifungal prescription. The efficacies of liposomal amphotericin B complex and anidulafungin for the treatment of pulmonary aspergillosis caused by different Aspergillus species need to be periodically evaluated in the future.

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Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): Thanks extended to the authors for carrying out this analysis. It is important to know the prevalences of resistance rates and antifungal susceptibility across the world. I have a few suggestions to improve the manuscript, which will improve the readability.
I find the introduction a bit disjointed -it starts with "septicemia can cause life-threatening complications", and then goes on to talk about invasive candidiasis and candidemia, and later invasive pulmonary aspergillosis, without linking back to septicemia at all. I do not agree that 'periodic monitoring of fungal species-specific susceptibility is beneficial for understanding the evolution of antifungal resistance'; whilst monitoring of susceptibility is needed, it tells you nothing about how resistance has evolved, or the mechanisms behind it. It does, however, tell you about the changes in prevalence temporally and spatially. This phrase of 'evolutionary changes' is mentioned again on page 8 when referring to Figure 1, which shows the changes in prevalence of susceptibility, and not the evolutionary changes responsible for this. Indeed, the legend of Figure 1 actually describes these data accurately, and this is how it should be referred to throughout the paper. Consider using 'Czechia' instead of the Czech Republic, as it wishes to be known by the former.
Pharmocokinetic profiles of the antifungal agents are only mentioned in the discussion; there is no analysis of these in relation to the findings in the results (in the introduction it is stated that "PK profiles" will be "assessed" -but they aren't. They're just provided in the discussion).
Also, the discussion mentions that single point alterations and other mutations were not identified in VRC-resistant A. fumigatus isolates -these data need to be included in the results, and also how they were identified included in the methods.
Reviewer #2 (Comments for the Author): This manuscript by Jean et al. summarized the susceptibility of Aspergillus and Candida isolates collected in clinics worldwide between 2017-2020. Studies that summarize susceptibility trends are important for monitoring the continued emergence of drug resistance among clinical isolates. The result presented here align well with previous studies from specific geographical regions however this study highlights interesting trends that have not previously reported. The following comments should be considered: 1. This study would be strengthened by defining the cutoffs used for determining susceptibility in tables 1-3 and subsequent figures. This will not only help readers who aren't familiar with specific cutoffs for each organism/drug but will also facilitate comparison with future studies where susceptibility cutoffs may change as more clinical outcome data is compiled for these treatments.
2. Lines 132-125 "During the four year study period..." The authors mention a wider fluctuation of AMB susceptibility than ISA or POSA and in figure 1 show much lower susceptibility in 2019 and 2020 compared to 2017 and 2018. It would be interesting to comment in the text on whether these trends are driven by large changes in certain regions or specific countries or if resistance patterns changed globally between these years.

A few grammatical changes
Line 74 "immunity" changed to "immune" Line 76 "prescriptions" changed to "prescription" Line 78 "virus" deleted Line 211 "one-doubling dilutions" changed to "two-fold" Line 363 "antifungal antibiotics" changed to "antifungals" Reviewer #3 (Comments for the Author): Jean and collaborators evaluated the in vitro antifungal susceptibilities against clinically important isolates of Aspergillus and Candida species in an antifungal resistance Surveillance program. The data obtained in this work will contribute to the epidemiology of resistance to antifungal agents.

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Reviewer #1 (Comments for the Author):
Thanks extended to the authors for carrying out this analysis. It is important to know the prevalences of resistance rates and antifungal susceptibility across the world. I have a few suggestions to improve the manuscript, which will improve the readability.
I find the introduction a bit disjointed -it starts with "septicemia can cause life-threatening complications", and then goes on to talk about invasive candidiasis and candidemia, and later invasive pulmonary aspergillosis, without linking back to septicemia at all.

Reply:
We have revised this sentence accordingly.
I do not agree that 'periodic monitoring of fungal species-specific susceptibility is beneficial for understanding the evolution of antifungal resistance'; whilst monitoring of susceptibility is needed, it tells you nothing about how resistance has evolved, or the mechanisms behind it. It does, however, tell you about the changes in prevalence temporally and spatially. This phrase of 'evolutionary changes' is mentioned again on page 8 when referring to Figure 1, which shows the changes in prevalence of susceptibility, and not the evolutionary changes responsible for this. Indeed, the legend of Figure 1 actually describes these data accurately, and this is how it should be referred to throughout the paper.

Reply:
We thank the reviewer's valuable suggestion. We have revised them accordingly.
Consider using 'Czechia' instead of the Czech Republic, as it wishes to be known by the former.

Reply:
We have revised them in the text and edited Figure 2 accordingly. Pharmacokinetic profiles of the antifungal agents are only mentioned in the discussion; there is no analysis of these in relation to the findings in the results (in the introduction it is stated that "PK profiles" will be "assessed" -but they aren't. They're just provided in the discussion).

Reply:
In fact, using the mathematical calculations according to the respective PK profile and required PD parameter(s), we have evaluated the suitability of 2 currently recommended antifungal doses against the tested intrapulmonary isolates of specific Aspergillus species by means of the MIC distributions and susceptibility data in the ATLAS that were presented in the original manuscript. Also, the discussion mentions that single point alterations and other mutations were not identified in VRC-resistant A. fumigatus isolates -these data need to be included in the results, and also how they were identified included in the methods.

Reply:
We apologized for being unable to provide the results regarding the genetic mutations in voriconazole (VRC)-resistant A. fumigatus isolates because these VRC-resistant isolates are not available for genetic analysis.
Consequently, we have revised the relevant statement in the Discussion section of the revised manuscript. Additionally, we will not add the methods employed for the analysis of resistance genes in intrapulmonary VRC-resistant A. fumigatus isolates in the revised manuscript as well.

Reviewer #2 (Comments for the Author):
This manuscript by Jean et al. summarized the susceptibility of Aspergillus and Candida isolates collected in clinics worldwide between 2017-2020. Studies that summarize susceptibility trends are important for monitoring the continued emergence of drug resistance among clinical isolates. The result presented here align well with previous studies from specific geographical regions; however, this study highlights interesting trends that have not previously reported. The following comments should be considered: 1. This study would be strengthened by defining the cutoffs used for determining susceptibility in tables 1-3 and subsequent figures. This will not only help readers who aren't familiar with specific cutoffs for each organism/drug but will also facilitate comparison with future studies where susceptibility cutoffs may change as more clinical outcome data is compiled for these treatments.
Reply: For strengthening the understanding of the in vitro susceptibility cutoff points of various antifungals, we decide to add a new Table (Table 4) which lists the susceptible breakpoints of different antifungals against various Aspergillus species and Candida species according to the CLSI 2020 and EUCAST 2020 guidelines in the revised manuscript.
2. Lines 132-125 "During the four year study period..." The authors mention a wider fluctuation of AMB susceptibility than ISA or POSA and in Figure 1 show much lower susceptibility in 2019 and 2020 compared to 2017 and 2018. It would be interesting to comment in the text on whether these trends are driven by large changes in certain regions or specific countries or if resistance patterns changed globally between these years.

Reply:
We indeed have no idea about this significant variation in annual AMB susceptibility rates against intrapulmonary A. fumigatus isolates. Hence, we have added this comment and relevant information regarding the decreased AMB susceptible rates in A. fumigatus which was observed by other investigators to the text of the revised manuscript.
3. A few grammatical changes Line 74 "immunity" changed to "immune" Line 76 "prescriptions" changed to "prescription" Line 78 "virus" deleted Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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